1. Field of the Invention
The present invention provides an apparatus and methods for the transformation of cells by electroporation. More particularly, the invention provides a ready-to-use cuvette for electroporation and methods of making and using such a cuvette.
2. Description of the Related Art
Introducing nucleic acids into cells is central to many types of biological experiments and biotechnology development methods. For example, when searching for a gene of interest in a cDNA library, the library must be transferred into a host organism. Among the various methods used for introducing nucleic acids into host cells, electroporation has gained widespread use. Exemplary methods and kits for performing electroporation are disclosed in U.S. Pat. Nos. 4,910,140 and 5,186,800 to Dower, and 6,338,965 to Greener et al., each of which is incorporated herein by reference in their entirety.
In general, electroporation involves the transfer of nucleic acids into a host cell by exposure of the cell to a high voltage electric impulse in the presence of the nucleic acids, such as genes or gene fragments. Typically, host cells are stored by freezing them at a temperature that preserves their viability for a long term. The frozen cells are stored in a separate container and must be defrosted, mixed with nucleic acid and subsequently transferred to a cuvette prior to electroporation.
An example of an electroporation method is disclosed in U.S. Pat. No. 5,186,800 and involves growing bacteria in enriched media (of any sort) and concentrating the bacteria by washing in a buffer containing 10% glycerol. DNA is added to the cells, the DNA and cells are mixed and the resulting mixture is subjected to an electrical discharge (pulse), which temporarily disrupts the outer cell wall of the bacterial cells and permits the DNA to enter the cells.
The efficiency of nucleic acid transfer depends on a variety of factors, including the electrical field strength, the pulse decay time, the pulse shape, the temperature in which the electroporation is conducted, the type of cell, the type of suspension buffer, and the concentration and size of the nucleic acid to be transferred. Researchers have modified the host cell suspension materials to aid in freezing the cells before the electrical treatment. Methods disclosed in U.S. Pat. No. 6,338,965 include the addition of sugars or sugar derivatives, e.g., sugar alcohols, to host cells suspended in a substantially non-ionic solution, either prior to initial freezing, or after thawing, but prior to electrotransformation, which improve electroporation efficiency.
Known methods of preparing frozen cells for electroporation require thawing the cells and mixing with nucleic acid prior to adding them to a suitable electroporation cuvette. This sequence of steps has always been deemed essential for at least two reasons: first, the structures of an electroporation cuvette are precisely dimensioned in order to provide reproducible electrical field strengths in the cell solution, and the freezing procedures necessary to store cells in the cuvette were considered too harsh to maintain these precise dimensions; second, the size of the cuvette chamber was considered too small to allow efficient mixing of cells with nucleic acid. Efficient mixing of cells and nucleic acid is essential to achieving a desired level of cell transformation by electroporation. The steps of thawing and mixing host cells prior to electroporation require experimenter's time and presents an opportunity for contamination or experimental errors that may impact results or diminish electrotransformation yields. Accordingly, there is a need for a method and equipment that will eliminate the need to separately thaw and prepare cells before placing them in an electroporation cuvette.